Palmitic acid (PA), impairs insulin sensitivity in skeletal muscle, and replacing PA in the diet with oleic acid (OA), a monounsaturated fatty acid (FA), may be beneficial. The first objective of this project is to understand the effects on lipid metabolism and skeletal muscle lipid composition, insulin signaling, and inflammatory signaling of two common variations in FA composition of the diet: (1) The typical intake of North America where PA and OA are present in equal proportions (HI PA diet). (2) The Mediterranean FA composition in which PA is much lower and OA much higher (HI OA diet). PA may induce insulin resistance in skeletal muscle cells via its accumulation in lipids within muscle cells and via activation of inflammatory signaling. The second objective of this project is to assess the hypothesis that a high intake of PA will down-regulate its own one-carbon (initial) oxidation, leading to increased inflammatory signaling and decreased insulin signaling. However, there is literature evidence that FA may induce defects in insulin signaling, if FA are not completely oxidized; therefore, the third objective is to assess the hypotheses that a high PA diet may decrease complete oxidation of FA and possibly accelerate initial FA oxidation. A double-masked, cross-over trial of the effects of a high PA diet versus a high OA/low PA diet in 16 overweight or obese subjects and 16 lean subjects (aged 18 - 40 yr) will be conducted to investigate the following Specific Aims: 1. To test the hypothesis that increased intake of PA will cause a decreased rate of [1-13C]-PA oxidation and will be associated with: (a) increased inflammatory signaling, within the muscle; (b) Decreased insulin signaling as characterized by decreased, whole body, peripheral insulin sensitivity (euglycemic/hyperinsulinemic clamp) and, in skeletal muscle, decreased phospho-AKT (Ser473), increased phospho-IRS-1 (Ser636/Ser639), decreased tyrosine phosphorylation of IRS-1, and decreased membrane content of GLUT4. 2. To test the hypothesis that increased intake of PA will cause less complete mitochondrial fatty acid oxidation, perhaps associated with dysfunction of the TCA cycle and increased reactive oxygen species formation. This hypothesis will be tested by measuring whole body and muscle (upper limb) relative rates of oxidation of [13-13C]-PA and [1-13C]-PA and by determining the serum profile of acylcarnitines, the urine concentrations of organic acids, and muscle concentrations of protein carbonyls. 3. To test the hypothesis that a high PA diet will lead to less complete oxidation of FA, less insulin signaling in skeletal muscle in response to a test meal, less whole body insulin sensitivity, increased dysfunction of the TCA cycle, and greater reactive oxygen species formation compared to the results obtained in obese versus lean humans. PUBLIC HEALTH RELEVANCE: High intakes of saturated fat are associated with diabetes. Our work has shown that the two most common fatty acids in the North American diet, palmitic acid (saturated fat) and oleic acid (monounsaturated fat) are metabolized differently and have opposite effects on fat burning. The proposed study will examine biochemical and molecular mechanisms for how a high saturated fat diet versus a low saturated fat/high monounsaturated fat diet alters the action of the hormone, insulin, in skeletal muscle.